Acinetobacter baumannii and A. nosocomialis are clinically relevant members of the Acinetobacter calcoaceticus-A. baumannii (Acb) complex and important opportunistic nosocomial pathogens (Wisplinghoff et al., 2012). These species have emerged as troublesome pathogens due in part to their remarkable resistance to disinfection, desiccation, as well as their ability to acquire multiply drug resistant phenotypes, all of which promote their survivability in the hospital setting. Furthermore, pan-resistant strains within the Acb are continuously being isolated from hospitals worldwide (Arroyo et al., 2009; Gottig et al., 2014). While the mechanisms of antibiotic resistance of Acb members has been intensively studied (Gordon et al., 2010), our understanding of their virulence mechanisms is unclear. Identified virulence factors include an outer membrane protein A (OmpA), the ability to form biofilms, exopolysaccharide, lipopolysaccharide (LPS), protein glycosylation systems and capsule (Choi et al., 2008; Choi et al., 2009; Gordon et al., 2010; Iwashkiw et al., 2012; Lees-Miller et al., 2013). A type VI secretion system (T6SS) has been also identified, although a role in pathogenesis has not been demonstrated (Carruthers et al., 2013; Weber et al., 2013).
A. baylyi is a non-pathogenic member of the genus Acinetobacter, characterized by its genetic tractability and natural competence. For these properties, A. baylyi is widely used as a model organism for molecular and genetic studies of the genus Acinetobacter (Vaneechoutte et al., 2006; de Berardinis et al., 2008; Brzoska et al., 2013) and is also utilized in bioremediation (Abd-El-Haleem et al., 2002; Mara et al., 2012). All members of the Acinetobacter genus, independent of their pathogenicity, carry a protein glycosylation system (Iwashkiw et al., 2012).
Protein glycosylation, the covalent attachment of carbohydrate moieties to protein substrates, is the most abundant post-translational modification of proteins (Varki, 1993) and occurs in all domains of life (Neuberger, 1938; Sleytr, 1975; Mescher & Strominger, 1976). The major types of protein glycosylation are N- and O-glycosylation. Both processes can be classified as oligosaccharyltransferase (OTase)-dependent and OTase independent (Nothaft & Szymanski, 2010; Iwashkiw et al., 2013). OTases are enzymes that catalyze the transfer of a glycan, previously assembled by cytoplasmic glycosyltransferases (GT) onto an undecaprenyl pyrophosphate lipid carrier, to target proteins. The development of sensitive analytical techniques has led to the identification of OTase-dependent protein glycosylation in numerous bacterial species. These include members of the genera Campylobacter, Neisseria, Pseudomonas, Francisella, Vibrio, Burkholderia and Bacteroides (Szymanski et al., 1999; Faridmoayer et al., 2007; Egge-Jacobsen et al., 2011; Balonova et al., 2012; Gebhart et al., 2012; Coyne et al., 2013; Lithgow et al., 2014). Glycosylation frequently affects protein stability, bacterial adhesion, flagellar filament assembly, biofilm formation, and virulence in general (Logan, 2006; Iwashkiw et al., 2013). An OTase-dependent, ubiquitous O-linked protein glycosylation system has been recently discovered within the genus Acinetobacter. This system was required for biofilm formation and pathogenicity of A. baumannii (Iwashkiw et al., 2012). The glycan structures for several strains of A. baumannii have also been characterized and extensive carbohydrate diversity has been established (Scott et al., 2014).
OTases involved in O-glycosylation (O-OTases) do not share extensive primary amino acid sequence homologies; yet, all O-Otases contain domains from the Wzy_C superfamily (Power and Jennings, 2003). Orthologs of PglL general 90 O-Otases and WaaL O-antigen ligases are two of the most well characterized enzymes from the Wzy_C superfamily. It has proven challenging to identify O-OTases based solely on bioinformatic methodologies as O-OTases and WaaL ligases catalyze similar reactions, i.e. the transfer of lipid-linked glycans to acceptor proteins or lipid A respectively (Hug & Feldman, 2011). The two enzymes appear to be evolutionarily and mechanistically related as mutagenesis of topologically similar conserved histidine residues of the E. coli O-antigen ligase (H337) and N. meningitidis O-OTase (H349) results in the loss of glycan transfer activities (Perez et al., 2008; Ruan et al., 2012; Musumeci et al., 2014). Recently, the PglL_A and PglL_B hidden Markov models (HMM) were defined to better resolve orthologs of PglL O-OTases from other enzymes of the Wzy_C superfamily (Power et al., 2006; Schulz et al., 2013).
O-OTases are often encoded downstream of their cognate target protein. This genetic arrangement is often found in Gram-negative organisms encoding type IV pili (Tfp) systems, where the major pilin subunit gene is immediately 5′ of the cognate OTase gene (Schulz et al., 2013). For example, in P. aeruginosa strain 1244 the major pilin, PilA, is glycosylated by PilO (later renamed TfpO), an O-OTase encoded immediately downstream of pilA (Castric, 1995; Kus et al., 2004). This modification is believed to play a role in virulence as glycosylation-deficient mutants showed decreased twitching motility and were out-competed by the wild type in a mouse respiratory infection model (Kus et al., 2004; Smedley et al., 2005). The same genetic arrangement and glycosylation phenotype has also been found in P. syringae (Nguyen et al., 2012).
Pilin post-translational modification has also been identified in Acinetobacter species. In A. baylyi ADP1, two Wzy_C superfamily domain-containing proteins are encoded in the genome. One gene is found immediately downstream of the gene encoding the pilin-like protein ComP, whereas the other gene is found within a distant glycan biosynthesis gene cluster. Mutation of the predicted OTase encoded downstream of the comP gene affected the electrophoretic mobility of ComP, indicating this gene may encode for a ComP-specific OTase (Porstendorfer et al., 2000; Schulz et al., 2013). Additionally, during the course of a previous study demonstrating the functional production of Tfp by the medically relevant A. nosocomialis strain M2, we also identified two molecular forms of PilA differing by apparent molecular weight leading to the hypothesis that the pilins of Acb members may also be post-translationally modified (Harding et al., 2013; Carruthers et al., 2013).
OTases are powerful tool for glycoengineering conjugate vaccines. The enzymatic attachment of glycans to proteins present several advantages compared to the chemical attachment of sugars. Although they exhibit relaxed specificity, OTases known so far present some limitations. For example, glycans containining glucose at the reducing end have not been successfully transferred by any of the known enzymes, such as PglB and PglL. PglB has been shown to required an acetylated sugar at the reducing end (Wacker et al., 2006). PglL was able to transfer sugars with galactose at the reducing end (Faridmoayer et al., 2008), but it has not been shown that sugars containing a glucose at the reducing can be transferred to proteins. This is extremely important for the synthesis of vaccines against Streptococcus. Most capsular polyccharides from Streptococcus contain a glucose residue at the reducing end (Bentley et al., 2006). The licensed vaccines against S. pneumoniae, such as Prevnar 13, contain up to 13 capsular serotypes. Better vaccines, containing more serotypes are needed. The current OTases have not been useful in generating conjugates containing capsule from these bacteria containing glucose at the reducing end, and therefore they have little applications for production of vaccines against Streptococcus. 
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.